This project has succeeded in determining the structures of three allergens, two from house dust mites, and one from peanuts in the past year. The significance of each project is discussed below. Der p 7- More than 50% of house dust mite allergic patients react specifically to the protein Der p 7. The natural function of many house dust mite allergens is thought to be related to the generation of the allergic response. However, the function of Der p 7 could not be determined from sequence information alone. The structure of Der p 7 determined by X-ray crystallography revealed an elongated structure with two 4-stranded beta sheets that wrap around a long C-terminal helix. This fold is highly similar to human proteins involved in the human innate immune response to bacteria. We demonstrated that Der p 7 could bind to a bacterial derived compound from gram positive bacteria, which suggests that Der p 7 could skew the normal human response by interfering with the recognition of foreign lipid-like compounds. Therefore it would be very interesting to learn exactly what types of compounds Der p 7 binds. Our current efforts are focused on screening compounds using NMR that are suggested by the literature, and via computer simulations. Knowing the class of ligands should provide more insight as to why the allergic response is so potent for dust mite allergens and Der p 7. Der p 5- In tropical climates such as Florida and Peurto Rico, the group 5 allergen Blo t 5 is a major allergen, while group 5 allergens from Dermatophagoides spp. in other parts of the United States are less important. The structure of Der p 5 was determined by X-ray crystallography to better understand this discrepancy, to investigate the biologic function in mites, and for comparison to two conflicting structures of Blo t 5. Der p 5 is a three helix bundle similar to the Blo t 5 structures, but the comparisons reveal that one structure is likely incorrect. Der p 5 differs from Blo t 5 in that the small angle X-ray scattering (SAXS) suggests Der p 5 is multimeric in solution while Blo t 5 is monomeric. This may be related to the prevalence of the immune response in different geographic regions. The Der p 5 crystal structure revealed a mechanism whereby Der p 5 can form polymers, which may be related to previous electron microscopic immunostaining that showed Der p 5 was a structural protein in mites located in fibrous structures. The structure also revealed a large hydrophobic cavity, which has the potential to bind hydrophobic ligands similar to Der p 7 and other allergens. Current efforts to identify a ligand are proceeding along the same lines as Der p 7 listed above using NMR binding assays guided by literature suggestions and in silico screening. Hydrophobic ligand binding is a common feature of many allergens. Ara h 2- The protein Ara h 2 is the most potent peanut allergen recognized by >90% of peanut allergic patients. However, the structure of this important allergen has not previously been determined. The structure is a 5 helix bundle held together by 4 disulfide bonds and related to the prolamin protein superfamily. The fold is highly similar to the related Ara h 6, but significant differences in the structures exist at the C-terminus covering approximately the surface area of one antibody epitope. Comparison of the two structures reveals differences in non-conserved residues that are surface exposed, which may explain differences in patient cross reactivity to the two peanut allergens. The data allow interpretation of previous biophysical and IgE antibody studies of Ara h 2. It is anticipated that the structure will provide information for the rational design of Ara h 2 hypoallergens for improved and safer immunotherapies.